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Characterization of IKAP/hELP1-dependent pathways/ Caractérisation des mécanismes moléculaires impliquant la protéine IKAP/hELP1 et le complexe ELongatorCornez, Isabelle 11 June 2008 (has links)
Characterization of IKAP/hELP1-dependent pathways.
Abstract
An extensive characterization of the signal transduction pathways is required to better understand how cells respond to various stimuli. While the human genome is completely sequenced, it is still necessary to combine those informations with a full knowledge of the biological roles played by the proteins. Importantly, a deregulation of the signal transduction pathways underlie a variety of human diseases such as cancer or neurodegenerative disorders.
IKAP/hELP1 is the largest subunit of Elongator and is required for the functional integrity of this complex. The histone acetyltransferase activity of Elongator helps the transcriptional machinery to move on the template of still poorly characterized genes to transcribe. In yeast, Elongator has also been involved in tRNA modifications as well as in exocytosis. In humans, Familial dysautonomia, an autosomal recessive disease characterized by defects in the development and maintenance of neurons of the autonomic and sensory systems, results from a loss-of-function of IKAP/hELP1. Recent work in our laboratory linked this disease to a cell migration defect of IKAP/hELP1 depleted cells.
The aim of this work is to investigate the role of Elongator in signal transduction. In the first part, we wanted to know whether Elongator was required for the regulation of gene expression in response to DNA damage. We demonstrated that some p53-dependent genes were aberrantly expressed upon Elongator deficiency in colon cancer-derived cells. Moreover, we showed that these IKAP/hELP1 depleted cells were not more sensitive to apoptosis in response to persistent DNAdamage.
In the second part of this work, to better characterize IKAP/hELP1, we tried to validate its potential interaction with the RanBP2 nucleoporin which is a component of the nuclear pore complex. Given its mainly cytoplasmic localization and its role in the nucleus, we studied the translocation of IKAP/hELP1 between both of these cellular compartments. We determined a potential nuclear export signal on the C-terminal part of IKAP/hELP1. This might allow us to further explore the link between the distinct roles and the localization of the Elongator complex. /
Caractérisation des mécanismes moléculaires impliquant la protéine IKAP/hELP1 et le complexe Elongator.
Résumé
Létude des voies de signalisation permet de mieux comprendre comment une cellule réagit face à divers stimuli. Alors que le génome humain est complètement séquencé, la caractérisation des différentes voies de signalisation cellulaire est à ce jour toujours incomplète et nécessite une meilleure connaissance de leurs principaux acteurs, les protéines. Ceci est dautant plus important quun dérèglement de lactivation de ces voies de signalisation peut conduire à des pathologies aussi diverses que le cancer ou des maladies neurodégénératives.
IKAP/hELP1 est la plus grande sous-unité du complexe Elongator et est essentielle pour lassemblage fonctionnel de celui-ci. Le complexe Elongator grâce à son activité dacétylation des histones qui permet laccès à la chromatine, participe à lélongation de la transcription de gènes, ceux-ci restant à ce jour peu caractérisés. Récemment chez la levure, Elongator a été décrit comme prenant part à dautres évènements cellulaires aussi divers que la modification des ARNs de transfert qui permet une traduction fidèle des protéines, ou que lexocytose. Chez lhomme, une neuropathologie génétique, la dysautonomie familiale, est la conséquence directe dune perte de fonction de la protéine IKAP/hELP1. Notre laboratoire a récemment fait le lien entre cette maladie qui affecte le développement et la survie du système nerveux autonome et sensoriel, et un déficit des capacités migratoires de cellules exprimant trop faiblement la protéine IKAP/hELP1.
Le but de ce travail est de poursuivre la caractérisation des rôles biologiques du complexe Elongator. Dans la première partie de ce travail, nous avons examiné le rôle dElongator dans la modulation de lexpression de gènes suite à un dommage à lADN. Nous avons pu mettre en évidence une altération du profil dexpression de plusieurs gènes connus pour être sous la dépendance de p53, une protéine activée en réponse à divers signaux de stress, dans des cellules dérivées de cancers du colon et déficientes pour Elongator. De plus, nous avons déterminé que ces cellules déplétées pour IKAP/hELP1 nétaient pas plus sensibles à lapoptose, en réponse ou non à des dommages persistants à lADN.
Dans la deuxième partie, nous avons tenté de valider, sur base de résultats obtenus chez la levure, son interaction potentielle avec une protéine du pore nucléaire, RanBP2. Etant donné sa localisation majoritairement cytoplasmique et sa fonction dans le compartiment nucléaire, nous avons étudié le transport dIKAP/hELP1 entre le noyau et le cytoplasme. Nous avons pu déterminer un site dexport nucléaire potentiel sur lextrémité C-terminale dIKAP/hELP1 qui nous permettra dexplorer le lien entre les différentes fonctions et la localisation du complexe Elongator.
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Two Types of Fibrils in ATTR Amyloidosis : Implications for Clinical Phenotype and Treatment OutcomeIhse, Elisabet January 2011 (has links)
Systemic amyloidoses are a group of lethal diseases where proteins aggregate into fibrillar structures, called amyloid fibrils, that deposits throughout the body. Transthyretin (TTR) causes one type of amyloidosis, in which the aggregates mainly infiltrate nervous and cardiac tissue. Almost a hundred different mutations in the TTR gene are known to trigger the disease, but wild-type (wt) TTR is also incorporated into the fibrils, and may alone form amyloid. Patients with the TTRV30M mutation show, for unknown reasons, two clinical phenotypes. Some have an early onset of disease without cardiomyopathy while others have a late onset and cardiomyopathy. It has previously been described that amyloid fibrils formed from TTRV30M can have two different compositions; either with truncated molecules beside full-length TTR (type A) or only-full-length molecules (type B). In this thesis, the clinical importance of the two types of amyloid fibrils was investigated. We found that the fibril composition types are correlated to the two clinical phenotypes seen among TTRV30M patients, with type A fibrils present in late onset patients and type B fibrils in early onset patients. The only treatment for hereditary TTR amyloidosis has been liver transplantation, whereby the liver producing the mutant TTR is replaced by an organ only producing wt protein. However, in some patients, cardiac symptoms progress post-transplantationally. We demonstrated that the propensity to incorporate wtTTR differs between fibril types and tissue types in TTRV30M patients, with cardiac amyloid of type A having the highest tendency. This offers an explanation to why particularly cardiac amyloidosis develops after transplantation, and suggests which patients that are at risk for such development. By examining patients with other mutations than TTRV30M, we showed that, in contrast to the general belief, a fibril composition with truncated TTR is very common and might even be the general rule. This may explain why TTRV30M patients often have a better outcome after liver transplantation than patients with other mutations. In conclusion, this thesis has contributed with one piece to the puzzle of understanding the differences in clinical phenotype and treatment response between TTR amyloidosis patients, by demonstrating corresponding differences at a molecular level.
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Inhibition of TTR aggregation-induced cell death : a new role for serum amyloid P componentAndersson, Karin, Pokrzywa, M, Dacklin, Ingrid, Lundgren, Erik January 2013 (has links)
BACKGROUND: Serum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure. CONCLUSIONS/SIGNIFICANCE: Together, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated. / <p>Epub 2013 Feb 4.</p>
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Identifying Susceptibility Genes for Familial Pancreatic Cancer Using Novel High-resolution Genome Interrogation PlatformsAl-Sukhni, Wigdan 06 December 2012 (has links)
Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.
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Inhibition of Transthyretin Fibrillogenesis Using a Conformation Specific AntibodyBugyei-Twum, Antoinette 21 March 2012 (has links)
Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.
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Inhibition of Transthyretin Fibrillogenesis Using a Conformation Specific AntibodyBugyei-Twum, Antoinette 21 March 2012 (has links)
Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.
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Identifying Susceptibility Genes for Familial Pancreatic Cancer Using Novel High-resolution Genome Interrogation PlatformsAl-Sukhni, Wigdan 06 December 2012 (has links)
Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.
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Anxiety disorders in mothers and their children: prospective longitudinal community studySchreier, Andrea, Wittchen, Hans-Ulrich, Höfler, Michael, Lieb, Roselind 15 August 2013 (has links) (PDF)
The relationship between DSM-IV anxiety disorders and their clinical characteristics in mothers and anxiety in offspring was examined in 933 mother-child pairs from a longitudinal community study. Offspring of mothers with an anxiety disorder had an elevated risk of developing any anxiety disorder, compared with offspring of mothers with no anxiety disorder. Increased risk of anxiety in the offspring was especially associated with maternal social phobia and generalised anxiety disorder, and with maternal diagnoses of early onset, greater number and more severe impairment. These results suggest that the type of maternal anxiety disorder and its severity of manifestation contribute to mother-offspring aggregation of anxiety.
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L'estimation des facteurs d'équivalence de revenu : le cas du QuébecLavigne, Benoît January 2007 (has links) (PDF)
L'objectif de cet ouvrage est de procéder à l'estimation d'une échelle d'équivalence de revenu pour le Québec. Cet objectif revêt une importance particulière puisqu'une telle échelle semble ne jamais avoir été estimée pour le cas particulier du Québec. La méthode choisie repose sur l'estimation d'un système de demande complet tout en assurant l'exactitude des facteurs d'équivalence. Cette méthode s'inspire des travaux réalisés par Shelley A. Phipps (1998) sur les familles canadiennes, mais en modifiant le modèle de manière à admettre tous les types de ménages.
Les microdonnées utilisées proviennent de l'Enquête sur les dépenses des ménages de 1997 à 2004 réalisée par Statistique Canada. Les prix proviennent des indices des prix à la consommation annuels de 1997 à 2004, par province, tirés du tableau 326-0002 de Statistique Canada. Sept échelles d'équivalence, dont trois québécoises, sont obtenues à l'aide de sous-ensembles différents définis par des régions et des ensembles de biens différents. Nous avons débuté par utiliser cinq catégories de biens pour les régions du Québec, du Québec urbain, des Maritimes, des Prairies, de la Colombie-Britannique et de l'Ontario, puis, afin de comparer nos résultats avec les études précédentes, nous avons utilisé seulement quatre catégories de biens pour le Québec urbain. Les résultats obtenus permettent d'énoncer certains constats empiriques importants, notamment en ce qui a trait aux divergences rurales et urbaines, aux divergences interprovinciales et aux impacts du choix des biens pris en compte lors de l'estimation. Les échelles obtenues sont comparées à l'échelle implicite suivie par le gouvernement via ses programmes sociaux. On constate un écart considérable entre celles-ci. On constate également une disproportion adultes-enfants. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Facteurs d'équivalence, Échelles d'équivalence, Économie du bien-être, Revenu des ménages.
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Clinical and Epidemiological Studies of Wegener´s GranulomatosisKnight, Ann January 2007 (has links)
Wegener´s granulomatosis (WG) is an unusual, serious, systemic vasculitis with specific clinical findings. The studies in this thesis aim at broadening our understanding of the aetiology and outcome of WG. Patients with WG were identified in the In-patient Register 1975-2001. During this time the incidence increased three-fold, and neither ANCA-related increased awareness, nor diagnostic drift, seem to fully explain this trend, but it is still unclear if a true rise in incidence exists. Anti- neutrophil cytoplasmic antibodies (ANCA) have been presented as highly specific for vasculitis. In a series of consecutive cANCA/PR3-ANCA positive patients, we investigated the positive predictive value for ANCA, and the outcome of patients with a positive cANCA/PR3-ANCA but not vasculitis. These patients have a low future risk of developing vasculitis, possibly indicating that ANCA, in this setting, reflects neutrophil activating properties not specific to vasculitis. By linkage of the WG-cohort, and randomly selected population controls, to the Multi-generation register, we identified all first-degree relatives and spouses of patients and controls, totally encompassing some 2,000 patients and 70,000 relatives. Familial aggregation of WG was the exception, with absolute risks of < 1 per 1000.However, relative risks in first-grade relatives amounted to 1.56 (95% CI 0.35-6.90) such that a moderate familial aggregation cannot be excluded. In the WG-cohort, cancer occurrence and risk was compared to that of the general population. Patients with WG have an overall doubled risk of cancer, with particularly increased risks of bladder-cancer, haematopoietic cancers including lymphomas and squamous skin-cancer. In a case-control study nested within the WG-cohort, treatment with cyclophosphamide was compared among bladder-cancer patients and matched cancer-free controls. Absolute risk of bladder cancer as high as 10% some years after diagnosis were found, and this risk can partly be attributed to cyclophosphamide-treatment, with a dose-response relationship.
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